The cryptochromes

Cryptochromes are photoreceptors that regulate entrainment by light of the circadian clock in plants and animals. They also act as integral parts of the central circadian oscillator in animal brains and as receptors controlling photomorphogenesis in response to blue or ultraviolet (UV-A) light in plants. Cryptochromes are probably the evolutionary descendents of DNA photolyases, which are light-activated DNA-repair enzymes, and are classified into three groups - plant cryptochromes, animal cryptochromes, and CRY-DASH proteins. Cryptochromes and photolyases have similar three-dimensional structures, characterized by an α/β domain and a helical domain. The structure also includes a chromophore, flavin adenine dinucleotide (FAD). The FAD-access cavity of the helical domain is the catalytic site of photolyases, and it is predicted also to be important in the mechanism of cryptochromes.     

Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity

The circadian clock is entrained to the daily cycle of day and night by light signals at dawn and dusk. Plants make use of both the phytochrome (phy) and cryptochrome (cry) families of photoreceptors in gathering information about the light environment for setting the clock. We demonstrate that the phytochromes phyA, phyB, phyD, and phyE act as photoreceptors in red light input to the clock and that phyA and the cryptochromes cry1 and cry2 act as photoreceptors in blue light input. phyA and phyB act additively in red light input to the clock, whereas cry1 and cry2 act redundantly in blue light input. In addition to the action of cry1 as a photoreceptor that mediates blue light input into the clock, we demonstrate a requirement of cry1 for phyA signaling to the clock in both red and blue light. Importantly, Arabidopsis cry1 cry2 double mutants still show robust rhythmicity, indicating that cryptochromes do not form a part of the central circadian oscillator in plants as they do in mammals.     

A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides

Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the α-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity ( K _d∼10⁻⁸ M) but double-stranded DNA and single-stranded RNA with far lower affinity ( K _d∼10⁻⁶ M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides . Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.     

A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.     

动物隐花色素研究进展

动物隐花色素(Cryptochrome)分为I型和II型,对生物钟的调控作用广为人知。I型隐花色素可以感受光信号而介导转录抑制物降解,II型隐花色素不需感受光而直接充当转录抑制物。近期研究发现,动物隐花色素还参与免疫应答和糖代谢,并为果蝇等动物光信号诱导的化学磁感知所必需。对动物隐花色素的进一步研究将增加对动物感知磁场过程的了解,也将帮助开发针对糖尿病等疾病的干预方法。文章重点综述了动物隐花色素的克隆与表达、结构特征、生理功能及作用机制,为这一领域的研究提供参考。     

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue‐light receptors such as aureochromes to absorb shorter‐wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV‐HK) that functions as a new class of eukaryotic blue‐light receptor in the pico‐phytoplanktonic cell Ostreococcus tauri. This LOV‐HK is related to the large family of LOV‐HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV‐HKs, including O. tauri LOV‐HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV‐HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV‐HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV‐HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV‐HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV‐HK alters CCA1 rhythmicity under constant red light, irrespective of LOV‐HK’s photochemical reactivity, suggesting that O. tauri LOV‐HK also participates in regulation of the circadian clock independent of its blue‐light‐sensing property. Molecular characterization of O. tauri LOV‐HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.     

隐光敏素及其信号传导研究进展

隐光敏素是一种对植物生长发育起调节作用的与光敏素功能相似的蓝光受体。90’s后特别是近年来对植物隐光敏素进行了较深入的研究,已知隐光敏素存在于植物,也存在于动物中。隐光敏素在植物种子萌发中的去黄化作用、光周期诱导开花、调节昼夜节律性等方面起重要作用;本文介绍了隐光敏素基因及其蛋白质的特征特性,包括隐光敏素的结构特征,在植物中的分布、在细胞中定位及其基因表达情况; 在其基础上,概述了隐光敏素调节植物光形态建成和动、植物昼夜节律性过程中所起的作用,通过分析隐光敏素及其与互作蛋白的关系, 初步阐述了隐光敏素如何经过信号传导通道上的蛋白而起调节植物生命活动的功能。指出深入研究隐光敏素及其信号传导以阐明植物的光形态建成具有重大理论和实践意义。     

Evolutionary History of the Photolyase/Cryptochrome Superfamily in Eukaryotes

Background

Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth.

Results

We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6–4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ω = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000–541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth’s geological history.     

Role of structural plasticity in signal transduction by the cryptochrome blue-light photoreceptor

Cryptochromes are blue-light photoreceptors that regulate a variety of responses such as growth and circadian rhythms in organisms ranging from bacteria to humans. Cryptochromes share a high level of sequence identity with the light-activated DNA repair enzyme photolyase. Photolyase uses energy from blue light to repair UV-induced photoproducts in DNA through cyclic electron transfer between the catalytic flavin adenine dinucleotide cofactor and the damaged DNA. Cryptochromes lack DNA repair activity, and their mechanism of signal transduction is not known. It is hypothesized that a light-dependent signaling state in cryptochromes is created as a result of an intramolecular redox reaction, resulting in conformational rearrangement and effector binding. Plant and animal cryptochromes possess 30-250 amino acid carboxy-terminal extensions beyond the photolyase-homology region that have been shown to mediate phototransduction. We analyzed the structures of C-terminal domains from an animal and a plant cryptochrome by computational, biophysical, and biochemical methods and found these domains to be intrinsically unstructured. We show that the photolyase-homology region interacts with the C-terminal domain, inducing stable tertiary structure in the C-terminal domain. Importantly, we demonstrate a light-dependent conformational change in the C-terminal domain of Arabidopsis Cry1. Collectively, these findings provide the first biochemical evidence for the proposed conformational rearrangement of cryptochromes upon light exposure.     

Cryptochromes: enabling plants and animals to determine circadian time

Cryptochromes are flavin-containing blue light photoreceptors related to photolyases-they are found in both plants and animals and have recently been described for bacteria. In plants, cryptochromes perform a variety of functions including the entrainment of circadian rhythms. They serve a similar role in Drosophila and mammals, where the cryptochromes also perform an additional function as an essential component of the circadian clock.     

Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures

Circadian clocks exhibit ‘temperature compensation’, meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature‐dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature‐compensated clock model by adding passive temperature effects into only the light‐sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature‐dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems‐level understanding of period control in the plant circadian oscillator.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.     

Plant dual‐specificity tyrosine phosphorylation‐regulated kinase optimizes light‐regulated growth and development in Arabidopsis

Light controls vegetative and reproductive development of plants. For a plant, sensing the light input properly ensures coordination with the ever‐changing environment. Previously, we found that LIGHT‐REGULATED WD1 (LWD1) and LWD2 regulate the circadian clock and photoperiodic flowering. Here, we identified Arabidopsis YET ANOTHER KINASE1 (AtYAK1), an evolutionarily conserved protein and a member of dual‐specificity tyrosine phosphorylation‐regulated kinases (DYRKs), as an interacting protein of LWDs. Our study revealed that AtYAK1 is an important regulator for various light responses, including the circadian clock, photomorphogenesis and reproductive development. AtYAK1 could antagonize the function of LWDs in regulating the circadian clock and photoperiodic flowering. By examining phenotypes of atyak1, we found that AtYAK1 regulated light‐induced period‐length shortening and photomorphogenic development. Moreover, AtYAK1 mediated plant fertility especially under inferior light conditions including low light and short‐day length. This study discloses a new regulator connecting environmental light to plant growth.     

Focusing on the nuclear and subnuclear dynamics of light and circadian signalling

Circadian clocks provide organisms the ability to synchronize their internal physiological responses with the external environment. This process, termed entrainment, occurs through the perception of internal and external stimuli. As with other organisms, in plants, the perception of light is a critical for the entrainment and sustainment of circadian rhythms. Red, blue, far‐red, and UV‐B light are perceived by the oscillator through the activity of photoreceptors. Four classes of photoreceptors signal to the oscillator: phytochromes, cryptochromes, UVR8, and LOV‐KELCH domain proteins. In most cases, these photoreceptors localize to the nucleus in response to light and can associate to subnuclear structures to initiate downstream signalling. In this review, we will highlight the recent advances made in understanding the mechanisms facilitating the nuclear and subnuclear localization of photoreceptors and the role these subnuclear bodies have in photoreceptor signalling, including to the oscillator. We will also highlight recent progress that has been made in understanding the regulation of the nuclear and subnuclear localization of components of the plant circadian clock.